1. Field of the Invention
The present invention relates to a substantially pure thermostable DNA polymerase. Specifically, the DNA polymerase of the present invention is a Thermotoga DNA polymerase and more specifically a Thermotoga neapolitana (Tne) DNA polymerase or Thermotoga maritima (Tma) DNA polymerase. Preferably, the polymerase has a molecular weight of about 100 kilodaltons. The present invention also relates to the cloning and expression of the Thermotoga DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to hosts which express said genes. The DNA polymerase of the present invention may be used in DNA sequencing, amplification reactions, and cDNA synthesis.
This invention also relates to mutants of the Thermotoga DNA polymerase, including Tne and Tma DNA polymerase. Specifically, the DNA polymerases of the present invention have mutations which substantially reduce 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity; mutations resulting in the ability of the mutant DNA polymerase to incorporate dideoxynucleotides into a DNA molecule about as efficiently as deoxynucleotides; and mutations which substantially reduce 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity. The Thermotoga (e.g., Tne and Tma) mutant DNA polymerase of this invention can have one or more of these properties. These DNA polymerase mutants may also be used in DNA sequencing, amplification reactions, and cDNA synthesis.
The present invention is also directed to novel mutants of other DNA polymerases which have substantially reduced 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity.
DNA polymerases synthesize the formation of DNA molecules which are complementary to a DNA template. Upon hybridization of a primer to the single-stranded DNA template, polymerases synthesize DNA in the 5xe2x80x2 to 3xe2x80x2 direction, successively adding nucleotides to the 3xe2x80x2-hydroxyl group of the growing strand. Thus, in the presence of deoxyribonucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded DNA template, can be synthesized.
A number of DNA polymerases have been isolated from mesophilic microorganisms such as E. coli. A number of these mesophilic DNA polymerases have also been cloned. Lin et al. cloned and expressed T4 DNA polymerase in E. coli (Proc. Natl. Acad. Sci. USA 84:7000-7004 (1987)). Tabor et al. (U.S. Pat. No. 4,795,699) describes a cloned T7 DNA polymerase, while Minkley et al. (J. Biol. Chem. 259:10386-10392 (1984)) and Chatterjee (U.S. Pat. No. 5,047,342) described E. coli DNA polymerase I and the cloning of T5 DNA polymerase, respectively.
Although DNA polymerases from thermophiles are known, relatively little investigation has been done to isolate and even clone these enzymes. Chien et al., J. Bacteriol. 127:1550-1557 (1976) describe a purification scheme for obtaining a polymerase from Thermus aquaticus (Taq). The resulting protein had a molecular weight of about 63,000 daltons by gel filtration analysis and 68,000 daltons by sucrose gradient centrifugation. Kaledin et al., Biokhymiya 45:644-51 (1980) disclosed a purification procedure for isolating DNA polymerase from T. aquaticus YT1 strain. The purified enzyme was reported to be a 62,000 dalton monomeric protein. Gelfand et al. (U.S. Pat. No. 4,889,818) cloned a gene encoding a thermostable DNA polymerase from Thermus aquaticus. The molecular weight of this protein was found to be about 86,000 to 90,000 daltons.
Simpson et al. purified and partially characterized a thermostable DNA polymerase from a Thermotoga species (Biochem. Cell. Biol. 86:1292-1296 (1990)). The purified DNA polymerase isolated by Simpson et al. exhibited a molecular weight of 85,000 daltons as determined by SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. The enzyme exhibited half-lives of 3 minutes at 95xc2x0 C. and 60 minutes at 50xc2x0 C. in the absence of substrate and its pH optimum was in the range of pH 7.5 to 8.0. Triton X-100 appeared to enhance the thermostability of this enzyme. The strain used to obtain the thermostable DNA polymerase described by Simpson et al. was Thermotoga species strain FjSS3-B.1 (Hussar et al., FEMS Microbiology Letters 37:121-127 (1986)). Others have cloned and sequenced a thermostable DNA polymerase from Thermotoga maritima (U.S. Pat. No. 5,374,553, which is expressly incorporated herein by reference).
Other DNA polymerases have been isolated from thermophilic bacteria including Bacillus steraothermophilus (Stenesh et al., Biochim. Biophys. Acta 272:156-166 (1972); and Kaboev et al., J. Bacteriol. 145:21-26 (1981)) and several archaebacterial species (Rossi et al., System. Appl. Microbiol. 7:337-341 (1986); Klimczak et al., Biochemistry 25:4850-4855 (1986); and Elie et al., Eur. J. Biochem. 178:619-626 (1989)). The most extensively purified archaebacterial DNA polymerase had a reported half-life of 15 minutes at 87xc2x0 C. (Elie et al. (1989), supra). Innis et al., In PCR Protocol: A Guide To Methods and Amplification, Academic Press, Inc., San Diego (1990) noted that there are several extreme thermophilic eubacteria and archaebacteria that are capable of growth at very high temperatures (Bergquist et al., Biotech. Genet. Eng. Rev. 5:199-244 (1987); and Kelly et al., Biotechnol. Prog. 4:47-62 (1988)) and suggested that these organisms may contain very thermostable DNA polymerases.
In many of the known polymerases, the 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity is present in the N-terminal region of the polymerase. (Ollis, et al., Nature 313:762-766 (1985); Freemont et al., Proteins 1:66-73 (1986); Joyce, Cur. Opin. Struct. Biol. 1:123-129 (1991).) There are some ammo acids, the mutation of which are thought to impair the 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity of E. coli DNA polymerase I. (Gutman and Minton, Nucl. Acids Res. 21:4406-4407 (1993).) These amino acids include Tyr77, Gly103, Gly184, and Gly192 in E. coli DNA polymerase I. It is known that the 5xe2x80x2-exonuclease domain is dispensable. The best known example is the Klenow fragment of E. coli polymerase I. The Klenow fragment is a natural proteolytic fragment devoid of 5xe2x80x2-exonuclease activity (Joyce et. al., J. Biol. Chem. 257:1958-64 (1990).) Polymerases lacking this activity are useful for DNA sequencing.
Most DNA polymerases also contain a 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity. This exonuclease activity provides a proofreading ability to the DNA polymerase. A T5 DNA polymerase that lacks 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity is disclosed in U.S. Pat. No. 5,270,179. Polymerases lacking this activity are particularly useful for DNA sequencing.
The polymerase active site, including the dNTP binding domain is usually present at the carboxyl terminal region of the polymerase (Ollis et al., Nature 313:762-766 (1985); Freemont et al., Proteins 1:66-73 (1986)). It has been shown that Phe762 of E. coli polymerase I is one of the amino acids that directly interacts with the nucleotides (Joyce and Steitz, Ann. Rev. Biochem. 63:777-822 (1994); Astatke, J. Biol. Chem. 270:1945-54 (1995)). Converting this amino acid to a Tyr results in a mutant DNA polymerase that does not discriminate against dideoxynucleotides. See copending U.S. application Ser. No. 08/525,087, of Deb K. Chatterjee, filed Sep. 8, 1995, entitled xe2x80x9cMutant DNA Polymerases and the Use Thereof,xe2x80x9d which is expressly incorporated herein by reference.
Thus, there exists a need in the art to develop more thermostable DNA polymerases. There also exists a need in the art to obtain wild type or mutant DNA polymerases that are devoid of exonuclease activities and are non-discriminating against dideoxynucleotides.
The present invention satisfies these needs in the art by providing additional DNA polymerases useful in molecular biology. Specifically, this invention includes a thermostable DNA polymerase. Preferably, the polymerase has a molecular weight of about 100 kilodaltons. Specifically, the DNA polymerase of the invention is isolated from Thermotoga, and more specifically, the DNA polymerase is obtained from Thermotoga neapolitana (Tne) and Thermotoga maritima (Tma). The Thermotoga species preferred for isolating the DNA polymerase of the present invention was isolated from an African continental solfataric spring (Windberger et al., Arch. Microbiol. 151. 506-512, (1989)).
The Thermotoga DNA polymerases of the present invention are extremely thermostable, showing more than 50% of activity after being heated for 60 minutes at 90xc2x0 C. with or without detergent. Thus, the DNA polymerases of the present invention is more thermostable than Taq DNA polymerase.
The present invention is also directed to cloning a gene encoding a Thermotoga DNA polymerase enzyme. DNA molecules containing the Thermotoga DNA polymerase genes, according to the present invention, can be transformed and expressed in a host cell to produce the DNA polymerase. Any number of hosts may be used to express the Thermotoga DNA polymerase gene of the present invention; including prokaryotic and eukaryotic cells. Preferably, prokaryotic cells are used to express the DNA polymerase of the invention. The preferred prokaryotic host according to the present invention is E. coli. 
The present invention also relates mutant thermostable DNA polymerases of the Poll type and DNA coding therefor, wherein there is amino acid change in the O-helix which renders the polymerase nondiscriminatory against ddNTPs in sequencing reactions. The O-helix is defined as RXXXKXXXFXXXYX, wherein X is any amino acid.
The present invention also relates to Thermotoga DNA polymerase mutants that lack exonuclease activity and/or which are nondiscriminatory against ddNTPs in sequencing reactions.
The present invention is also directed generally to DNA polymerases that have mutations that result in substantially reduced or missing 5xe2x80x2xe2x86x923xe2x80x2 exonuclease activity.
In particular, the invention relates to a Thermotoga DNA polymerase mutant which is modified at least one way selected from the group consisting of
(a) to reduce or eliminate the 3xe2x80x2-5xe2x80x2 exonuclease activity of the polymerase;
(b) to reduce or eliminate the 5xe2x80x2-3xe2x80x2 exonuclease activity of the polymerase; and
(c) to reduce or eliminate discriminatory behavior against a dideoxynucleotide.
The invention also relates to a method of producing a DNA polymerase, said method comprising:
(a) culturing the host cell of the invention;
(b) expressing said gene; and
(c) isolating said DNA polymerase from said host cell.
The invention also relates to a method of synthesizing a double-stranded DNA molecule comprising:
(a) hybridizing a primer to a first DNA molecule; and
(b) incubating said DNA molecule of step (a) in the presence of one or more deoxy- or dideoxyribonucleoside triphosphates and the DNA polymerase of the invention, under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule. Such deoxy- and dideoxyribonucleoside triphosphates include dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, 7-deaza-dATP, dUTP, ddATP, ddCTP, ddGTP, ddlTP, ddTTP, [xcex1-S]dATP, [xcex1-S]dTTP, [xcex1-S]dGTP, and [xcex1-S]dCTP.
The invention also relates to a method of sequencing a DNA molecule, comprising:
(a) hybridizing a primer to a first DNA molecule;
(b) contacting said DNA molecule of step (a) with deoxyribonucleoside triphosphates, the DNA polymerase of the invention, and a terminator nucleotide;
(c) incubating the mixture of step (b) under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule, wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 3xe2x80x2 termini; and
(d) separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said first DNA molecule can be determined. Such terminator nucleotides include ddTTP, ddATP, ddGTP, ddITP or ddCTP.
The invention also relates to a method for amplifying a double stranded DNA molecule, comprising:
(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3xe2x80x2-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3xe2x80x2-termini of the second strand of said DNA molecule;
(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of the DNA polymerase of the invention, under conditions such that a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand are synthesized;
(c) denaturing said first and third strand, and said second and fourth strands; and
(d) repeating steps (a) to (c) one or more times.
The invention also relates to a kit for sequencing a DNA molecule, comprising:
(a) a first container means comprising the DNA polymerase of the invention;
(b) a second container means comprising one or more dideoxyribonucleoside triphosphates; and
(c) a third container means comprising one or more deoxyribonucleoside triphosphates.
The invention also relates to a kit for amplifying a DNA molecule, comprising:
(a) a first container means comprising the DNA polymerase of the invention; and
(b) a second container means comprising one or more deoxyribonucleoside triphosphates.
The present invention also relates to a mutant DNA polymerase having substantially-reduced or eliminated 5xe2x80x2-3xe2x80x2 exonuclease activity, wherein at least one of the amino acids corresponding to Asp8, Glu112, Asp114, Asp115, Asp137, Asp139, Gly102, Gly187, or Gly195 of Tne DNA polymerase has been mutated.
The present invention also relates to a method of producing a mutant DNA polymerase having substantially reduced or eliminated 5xe2x80x2-3xe2x80x2 exonuclease activity, wherein at least one of the amino acids corresponding to Asp8, Glu112, Asp114, Asp115, Asp137, Asp139, Gly102, Gly187, or Gly195 of Tne DNA polymerase has been mutated, comprising:
(a) culturing the host cell of the invention;
(b) expressing the mutant DNA polymerase; and
(c) isolating said mutant DNA polymerase.